Protective vaccine based on Staphylococcus aureus SA2451 protein

ABSTRACT

The present invention relates to methods of inducing an immune response to  Staphylococcus  comprising administering a composition comprising an SA2451-related polypeptide from  Staphylococcus aureus  as well as derivatives or fragments thereof. The present invention also encompasses methods of treating and/or reducing the likelihood of a  Staphylococcus  infection by administering a composition comprising an SA2451-related polypeptide or an antibody that specifically binds to an SA2451 polypeptide, derivative or fragments thereof. Compositions administered in the methods of the invention can include one or more additional antigens. Compositions used to practice the methods of the invention are also encompassed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a §371 national stage application ofPCT/US2012/062019, international filing date of Oct. 26, 2012, whichclaims priority to U.S. Ser. No. 61/553,589, filed Oct. 31, 2011, whichis herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of inducing an immune responseto Staphylococcus using an SA2451 protein from Staphylococcus aureus aswell as derivatives or fragments thereof. The present invention alsorelates to a composition, particularly an S. aureus vaccine, comprisingan SA2451 polypeptide, derivative or fragment thereof, alone or incombination with one or more additional immunogens, capable of inducinga protective immune response.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “MRLIFD00051USPCT-SEQLIST-29APR2014.TXT”, creation date ofApr. 28, 2014, and a size of 13.0 KB. This sequence listing submittedvia EFS-Web is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Staphylococcus aureus is a bacterial pathogen responsible for a widerange of diseases and conditions, ranging from minor skin infections toserious life-threatening wound infections, bacteraemia, endocarditis,pneumonia, osteomyelitis and toxic shock syndrome. While S. aureuscommonly colonizes in the nose and skin of healthy humans, often causingonly minor infections (e.g., pimples, boils), it can also cause systemicinfections. Examples of diseases and conditions caused by S. aureusinclude bacteremia, infective endocarditis, folliculitis, furuncle,carbuncle, impetigo, bullous impetigo, cellulitis, botryomyosis, toxicshock syndrome, scalded skin syndrome, central nervous systeminfections, infective and inflammatory eye disease, osteomyelitis andother infections of joints and bones, and respiratory tract infections.(The Staphylococci in Human Disease, Crossley and Archer (eds.),Churchill Livingstone Inc. 1997; Archer, 1998, Clin. Infect. Dis.26:1179-1181.)

Staphylococcus aureus is a nosocomial as well as a community-acquiredpathogen which causes several diseases ranging from. See Lowy et al.,1998, N. Engl. J. Med. 339:520-32. The worldwide growing incidence ofstaphylococcal infections is strongly related to the increased use ofsurgical devices and a growing number of immunocompromised patients. Thesituation has become more serious since the increased use of antibioticsled to the emergence of methicillin-resistant S. aureus strains (MRSA).See Selvey et al., 2000, Infect. Control. Hosp. Epidemiol. 21:645-8;Peacock et al., 1980, Ann. Intern. Med. 93:526-32. Additionally, S.aureus isolates with reduced susceptibility to vancomycin, theantibiotic of choice against MRSA strains, were described in the lab aswell as the clinic See Tenover et al., 2001, Emerg. Infect. Dis.7:327-32; Tenover et al., 1998, J. Clin. Microbiol. 36:1020-7; Palazzoet al., 2005, J. Clin. Microbiol. 43:179-85. The rising emergence ofmultidrug-resistant staphylococci has led to a growing interest in thedevelopment of alternative approaches to prevent and treatstaphylococcal infections.

Information concerning S. aureus polypeptide sequences has been obtainedfrom sequencing the S. aureus genome. See Kuroda et al., 2001, Lancet357:1225-1240; Baba et al., 2000, Lancet 359:1819-1827; Gill et al.,2005, J. Bacteriol. 187:2426-2438 and European Patent Publication EP 0786 519. To some extent, bioinformatics has been employed in efforts tocharacterize polypeptide sequences obtained from genome sequencing. See,e.g., European Patent Publication EP 0 786 519 and U.S. Pat. No.6,593,114.

Techniques such as those involving display technology and sera frominfected patients have also been used in an effort to help identifygenes coding for potential antigens. See, e.g., InternationalPublication Nos. WO 01/98499 and WO 02/059148; and Etz et al., 2002,Proc. Natl. Acad. Sci. USA 99:6573-6578. Numerous staphylococcal surfaceproteins have been identified so far using recently adoptedtechnologies, like proteomics (see Brady et al., 2006, Infect. Immun.74:3415; Gatlin et al., 2006, Proteomics 6:1530; Pieper et al., 2006,Proteomics 6:4246; Vytvytska et al., 2002, Proteomics 2:580; Nandakumaret al., 2005, J. Proteome Res. 4:250) or protein selection methods basedon expression libraries (see Clarke et al., 2006, J. Infect. Dis.193:1098; Etz et al., 2002, Proc. Natl. Acad Sci. USA 99:6573-8;Weichhart et al., 2003, Infect. Immun. 71:4633; and Yang et al., 2006,Vaccine 24:1117).

Vaccines consisting of one or more antigenic determinants can provideprotection against lethal challenge with S. aureus in mice. SeeStranger-Jones et al., 2006, Proc. Natl. Acad. Sci. USA 103:16942-7 andKuklin et al., 2006, Infect. Immun. 74:2215. Recombinantly expressedpolypeptides can readily be made, purified, and formulated as vaccines.Additionally, recombinant proteins can be combined with additionalcomponents to make multicomponent polypeptide vaccines that induce aspectrum of immune responses. Despite this, there are no reportedprotein based vaccines for staphylococcal infections in humans oranimals to date. Thus, there remains a need for immunogens that canprovide protective immunity against Staphylococcal infections in humanand/or animals.

SUMMARY OF THE INVENTION

It is shown herein that SA2451-related polypeptides can provideprotective immunity against S. aureus infection in a relevant animalmodel system. Accordingly, one aspect of the invention provides acomposition comprising an immunologically effective amount of apolypeptide that is at least 95% identical to a SA2451 polypeptide(represented by SEQ ID NO:1) or a fragment of the polypeptide and apharmaceutically acceptable carrier. In embodiments of the invention,the polypeptide is not SEQ ID NO:1. In more preferred embodiments, thepolypeptide is at least 98% identical to SEQ ID NO:1.

Also provided by the invention are compositions as described above,which further comprise one or more additional S. aureus antigens or oneor more additional antigens from another Staph species, such a S.epidermidis. The compositions of the invention may further comprise anadjuvant.

The present invention is further related to a method of inducing aprotective immune response in a patient against an S. aureus infectioncomprising the steps of administering to the patient an immunologicallyeffective amount of any of the compositions or vaccines describedherein. The compositions are administered to a patient in need thereof,wherein the patient is human or a non-human mammal such as a cow. Insome embodiments of the methods described herein, the compositions aregiven to a patient who suffers from weakened immunity, has received aforeign body implant or is on renal dialysis.

Also provided by the invention is the use of a polypeptide that is atleast 95% identical to SEQ ID NO:1 or a fragment of the polypeptide inthe manufacture of a medicament for inducing a protective immuneresponse in a patient against S. aureus infection or the use of thecompositions herein for inducing protective immunity against S. aureus.

Further provided by the invention is a method of conferring passiveimmunity to S. aureus infection in a patient comprising administering tothe patient one or more antibodies that specifically bind to apolypeptide of SEQ ID NO:1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 show the nucleotide sequence (SEQ ID NO:3) of SA2451 from S.aureus strain COL.

FIGS. 2A-2B show the amino acid sequences of SA2451 (SEQ ID NO:1) andSA2451 with a carboxy-terminal His-tag (SEQ ID NO:2), which was added tofacilitate purification of the antigen.

FIG. 3 shows the results from two independent murine lethal challengeexperiments, as described in Example 3. Balb/c mice were immunized witheither SA2451 or BSA formulated on Merck aluminum adjuvant (MAA) (seeEXAMPLE 2) and were challenged with S. aureus Becker.

FIGS. 4A and 4B compare the survival curves from two independent murinelethal challenge experiments, as described in Example 3 (Experiments#MSA116 and MSA120). In each experiment, mice were immunized with either(a) His-tagged SA2451 adsorbed onto MAA or (b) BSA, which was alsoformulated with MAA, as described in Example 2, and challenged with S.aureus Becker.

FIG. 5 shows the results of an immunogenicity study in which rhesusmacaques were immunized intramuscularly with SA2451 formulated on MAA.The animals were challenged via the IV route with a sublethal dose of S.aureus Becker. The antibody response to the antigen was measured usingan ELISA technique with a Mesoscale instrument (see Example 4).

DETAILED DESCRIPTION OF THE INVENTION

As stated above, there is a need for the identification of S. aureusimmunogens that can provide protective immunity against Staphylococcalinfections. To that end, the present invention provides vaccines andmethods of use thereof based on SA2451, which is an ABC transporterlipoprotein from S. aureus (Gill et al., 2005, J. Bacteriol.187:2426-2438); known as glycine betaine/carnitine/choline transportsystem permease (ABC transporter protein), alternatively osmoprotectantbinding protein (OpuCC). The ABC transporter family are diversetransmembrane proteins that utilize the energy of ATP hydrolysis tocarry out certain biological processes including translocation ofvarious substrates across membranes. The common feature of all ABCtransporters is that they consist of two distinct domains, thetransmembrane domain (TMD) and the nucleotide-binding domain (NBD)(Higgins, 1992, Annu. Rev. Cell Biol. 8:67).

SA2451 was originally identified for use as a potential immunogen in aStaph-based vaccine using a genomics approach. A conserved motif forStaphylococcus aureus lipoproteins was used to search the S. aureusgenome database. Antigens with the conserved motif were selected forcloning. Without being bound by theory, it was thought that antigenspossessing the conserved motif could have one end inserted into thebacterial cell membrane, and the other end extending through the cellwall to be surface exposed; thus making them potential vaccine targets.

SA2451 related sequences are disclosed in EP Patent Publication No.786519, WO 02/094868, and US 2010/047267. However, no data is providedto demonstrate the usefulness of these sequences in a protective vaccineagainst S. aureus. In contrast, the inventors have shown herein that aHis-tagged-derivative of SA2451 (SEQ ID NO:2) is able to provideprotective immunity from challenge with S. aureus strain Becker in amurine model system; e.g. Balb/c mice. It is further shown herein thatSA2451-related sequences (e.g. SEQ ID NO:2) can induce an immuneresponse in rhesus monkeys. The vaccines of the present invention canprovide protection against infection with S. aureus strains, e.g. strainBecker or strain COL.

A S. aureus SA2451 gene sequence was cloned from S. aureus strain COLand expressed recombinantly in E. coli. The recombinant protein isimmunogenic in rodents and protects the animals from S. aureusinfection. As used herein, the term “SA2451” refers to a polypeptide ofSEQ ID NO:1 or a naturally occurring allelic variant or a homolog fromanother S. aureus strain. Examples of other strains of S. aureus includeBecker, MW2, N315 (see Kuroda et al., 2001, Lancet 357:1225), Newman,USA300 (see Diep et al., 2006, Lancet 367:731), MSA817, and Mu3. In oneembodiment, SA2451 is SEQ ID NO:1, which is from S. aureus strain COL.SA2451 may also be from another S. aureus strain. “SA2451-relatedsequences” refer to sequences that are highly identical to the SA2451provided in SEQ ID NO:1, or derivatives or fragments thereof.Embodiments of the invention provide pharmaceutical compositions thatcomprise an immunologically effective amount of a SA2451 immunogen and apharmaceutically acceptable carrier, wherein the SA2451 immunogen is notSEQ ID NO:1 and wherein the SA2451 immunogen is a polypeptide that is atleast 95% identical to SEQ ID NO:1. SA2451 immunogens useful in thepresent invention are further defined, infra.

In one embodiment, SA2451 polypeptides, derivatives or fragments thereofare used as a vaccine for the treatment and/or reducing the likelihoodof staphylococcal infections. Methods of the invention encompassadministering a composition comprising a vaccine of the invention to anon-human animal or human patient in need thereof to induce an immuneresponse. In a specific embodiment of the invention, one or moreadditional antigens are provided in the composition comprising anisolated SA2451 polypeptide, derivative or fragment thereof. In oneembodiment, the additional antigen is IsdB (also known as ORF0657) or aderivative or fragment thereof.

As used herein, the phrase “induce an immune response” refers to theability of a polypeptide, derivative, or fragment thereof to produce animmune response in a patient, preferably a mammal, to which it isadministered, wherein the response includes, but is not limited to, theproduction of elements, such as antibodies, which specifically bind S.aureus or said polypeptide, derivative or fragment thereof. The immuneresponse provides a protective effect against S. aureus infection,ameliorates at least one pathology associated with S. aureus infectionand/or reduces the likelihood that a patient will contract an S. aureusinfection. In a specific embodiment, the immune response inducesopsonophagocytic activity of human neutrophils for S. aureus.

As used herein, the phrase “an immunologically effective amount” refersto the amount of an immunogen that can induce a protective immuneresponse against S. aureus when administered to a patient. The amountshould be sufficient to significantly reduce the likelihood or severityof an S. aureus infection and/or the clinical manifestations thereof.Animal models known in the art can be used to assess the protectiveeffect of administration of immunogen. For example, a murine,lethal-challenge model (see, e.g., Thakker et al., 1998, Inf Immun66:5183-5189; Fattom et al., 1996, Inf Immun 64:1659-1665) and a rat,indwelling-catheter, sub-lethal challenge model (see, e.g., Ulphani etal., 1999, Lab Animal Sc. 49:283-287; Baddour et al., 1992, J Inf Dis165:749-53; Ebert et al., Human Vaccines 7(6): 1-9 (2011)) can be used.

In another embodiment, SA2451 polypeptides, derivatives or fragmentsthereof are used as a target for generating antibodies. These antibodiescan be administered to a patient for the treatment and/or reduction ofthe likelihood of staphylococcal infections due to passive immunity.

As used herein, the phrase “passive immunity” refers to the transfer ofactive humoral immunity in the form of antibodies. Passive immunityprovides immediate protective effect to the patient from the pathogenrecognized by the administered antibodies and/or ameliorates at leastone pathology associated with pathogen infection. However, the patientdoes not develop an immunological memory to the pathogen and thereforemust continue to receive the administered antibodies for protection fromthe pathogen to persist.

Embodiments of the invention also include one or more of the polypeptideimmunogens or compositions thereof, described herein, or a vaccinecomprising or consisting of said immunogens or compositions (i) for usein, (ii) for use as a medicament for, or (iii) for use in thepreparation of a medicament for: (a) therapy (e.g., of the human body);(b) medicine; (c) inhibition of S. aureus replication; (d) treatment orprophylaxis of infection by S. aureus; or, (e) treatment, prophylaxisof, or delay in the onset or progression of S. aureus-associateddisease(s), including, but not limited to: skin infections, woundinfections, bacteremia, endocarditis, pneumonia, osteomyelitis, toxicshock syndrome, infective endocarditis, folliculitis, furuncle,carbuncle, impetigo, bullous impetigo, cellulitis, botryomyosis, scaldedskin syndrome, central nervous system infections, infective andinflammatory eye disease, osteomyelitis and other infections of jointsand bones, and respiratory tract infections. The polypeptide immunogensof the invention are also useful for treatment, prophylaxis of, or delayin the onset or progression of S. aureus-associated disease common toanimals including: bovine mastitis, respiratory disease in swine,skeletal problems, and skin infections in companion animals such ashorses, dogs and cats. In these uses, the polypeptide immunogens,compositions thereof, and/or vaccines comprising or consisting of saidimmunogens or compositions can optionally be employed in combinationwith one or more anti-bacterial agents (e.g., anti-bacterial compounds;combination vaccines, described infra).

Polypeptides

The amino acid sequence of a wild type full length SA2451 from S. aureussubsp. aureus COL is SEQ ID NO:1. SEQ ID NO:1 as well as derivatives andfragments thereof can be used in the methods of the invention.Collectively, derivatives and fragments of SEQ ID NO:1 are termed“altered polypeptides” or “SA2451-related sequence”. SEQ ID NO:2 is aHis-tagged derivative of SEQ ID NO:1 which was shown herein to provideprotective immunity against S. aureus infections in animal modelsystems.

As used herein, the term “isolated” indicates a different form thanfound in nature. The different form of the polypeptide can be, forexample, a different purity than found in nature. In one embodiment, theterm refers to polypeptides that are substantially or essentially freefrom components that normally accompany it in its native state.

As used herein, the terms “purified” with regard to, for example, apolypeptide immunogen indicates the presence of such polypeptide in anenvironment lacking one or more other polypeptides with which it isnaturally associated and/or is represented by at least about 10% of thetotal protein present. In different embodiments, the purifiedpolypeptide represents at least about 50%, at least about 75%, at leastabout 95%, or at least about 99% of the total protein in a sample orpreparation.

As used herein, the term “fragment” refers to a continuous segment of anSA2451 polypeptide (i.e., SEQ ID NO:1) or derivatives thereof having atleast 5 amino acid residues, at least 10 amino acid residues, at least15 amino acid residues, or at least 20 amino acid residues and which isshorter than the full length SA2451 polypeptide. Preferably, fragmentswill comprise at least one antigenic determinant or epitopic region. Insome embodiments, a fragment of the invention will comprise a domain ofthe SA2451 polypeptide including, but not limited to, the extracellulardomain or T cell epitopes (either from the intracellular orextracellular portion of SA2451). One or more fragments comprising atleast one antigenic determinant or epitopic region may be fusedtogether.

As used herein, the terms “epitope” or “antigenic determinant” refer toa site on an antigen to which an antibody and/or T cell receptor binds.Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, and more usually, at least 5 or 8-10amino acids in a unique spatial conformation. Methods of determiningspatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance.

As used herein, the term “derivative” refers to a polypeptide having oneor more alterations, which can be changes in the amino acid sequence(including additions and deletions of amino acid residues) and/orchemical modifications. In preferred embodiments, the derivative is atleast 85%, at least 90%, at least 95%, at least 97%, at least 99%identical to the original sequence prior to alteration. In somepreferred embodiments, the polypeptide is not SEQ ID NO:1. In general,derivatives retain the activity of inducing a protective immuneresponse. In some embodiments, SA2451 or a fragment thereof has beenaltered to a derivative of the invention such that one or more epitopeshave been enhanced. Epitope enhancement improves the efficacy of apolypeptide to induce a protective immune response. Epitope enhancementcan be performed using different techniques such as those involvingalteration of anchor residues to improve peptide affinity for MHCmolecules and those that increase the affinity of the peptide-MHCcomplex for a T-cell receptor (Berzofsky et al., 2001, Nature Review1:209-219).

In one embodiment, a derivative is a polypeptide that has an amino acidsequence which differs from the base sequence from which it is derivedby one or more amino acid substitutions. Amino acid substitutions may beregarded as “conservative” where an amino is replaced with a differentamino acid with broadly similar properties. “Non-conservative”substitutions are where amino acids are replaced with amino acids of adifferent type. Broadly speaking, fewer non-conservative substitutionswill be possible without altering the biological activity of thepolypeptide. In some embodiments, no more than 12 amino acid residues,11 amino acid residues, 10 amino acid residues, 9 amino acid residues, 8amino acid residues, 7 amino acid residues, 6 amino acid residues, 5amino acid residues, 4 amino acid residues, 3 amino acid residues, 2amino acid residues, or 1 amino acid residue is/are substituted.

In another embodiment, a derivative is a polypeptide that has an aminoacid sequence which differs from the base sequence from which it isderived by having one or more amino acid deletions and/or additions inany combination. Deleted or added amino acids can be either contiguousor individual residues. In some embodiments, no more than 25 amino acidresidues, no more than 20 amino acid residues, no more than 15 aminoacid residues, no more than 12 amino acid residues, no more than 10amino acid residues, no more than 8 amino acid residues, no more than 7amino acid residues, no more than 6 amino acid residues, no more than 5amino acid residues, no more than 4 amino acid residues, no more than 3amino acid residues, no more than 2 amino acid residues, or no more than1 amino acid residue is/are deleted or added. In additional embodiments,domains of SA2412 including, but not limited to, the transmembranedomain, are deleted. Addition of amino acids may include fusion (eitherdirectly or via a linker) to at least one functional protein domainincluding, but not limited to, marker polypeptides, carrier polypeptides(including, but not limited to, OMPC, BSA, OVA, THY, KLH, tetanustoxoid, HbSAg, HBcAg, rotavirus capsid proteins, L1 protein of the humanpapilloma virus, diphtheria toxoid CRM197 protein, flagellin and HPV VLPespecially type 6, 11 and 16), polypeptides holding adjuvant propertiesor polypeptides that assist in purification. Additionally, it will beappreciated that the additional amino acid residues can be derived fromS. aureus or an unrelated source and may produce an immune responseeffective against S. aureus or another pathogen.

In another embodiment, a derivative is a polypeptide that has an aminoacid sequence which differs from the base sequence from which it isderived by having one or more chemical modifications of the protein.Chemical modifications include, but are not limited to, modification offunctional groups (such as alkylation, hydroxylation, phosphatation,thiolation, carboxilation and the like), incorporation of unnaturalamino acids and/or their derivatives during protein synthesis and theuse of crosslinkers and other methods which impose conformationalconstraints on the polypeptides.

Any method known in the art can be used to determine the degree ofdifference between SA2451 (e.g., SEQ ID NO:1) and a derivative. In oneembodiment, sequence identity is used to determine relatedness.Derivatives of the invention will be at least 85% identical, at least90% identical, at least 95% identical, at least 97% identical, at least99% identical to the base sequence (e.g., SEQ ID NO:1). The percentidentity is defined as the number of identical residues divided by thetotal number of residues and multiplied by 100. If sequences in thealignment are of different lengths (due to gaps or extensions), thelength of the longest sequence will be used in the calculation,representing the value for total length.

In another embodiment, hybridization is used to determine relatedness.Nucleic acids encoding derivatives of the invention will hybridize tonucleic acids encoding SA2451 (e.g., SEQ ID NO:3) under highly stringentconditions. Stringency of hybridization reactions is readilydeterminable by one of ordinary skill in the art, and generally is anempirical calculation dependent upon probe length, washing temperature,and salt concentration. In general, longer probes require highertemperatures for proper annealing, while shorter probes need lowertemperatures. Hybridization generally depends on the ability ofdenatured DNA to re-anneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature which can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.The Tm is the temperature (under defined ionic strength, pH and nucleicacid concentration) at which 50% of the probes complementary to thetarget sequence hybridize to the target sequence at equilibrium. Sincetarget sequences are generally present at excess, at Tm, 50% of theprobes are occupied at equilibrium. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0to 8.3; and the temperature is at least about 30° C. for short probes,primers or oligonucleotides (e.g., 10 to 50 nucleotides) and at leastabout 60° C. for longer probes, primers and oligonucleotides. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide. For additional details and explanation ofstringency of hybridization reactions, see Ausubel et al., CurrentProtocols in Molecular Biology, Wiley Interscience Publishers, (1995).

As used here, the phrase “high stringency” refers to conditions that:(1) employ low ionic strength and high temperature for washing, forexample 0.015 M sodium chloride/0.0015 M sodium citrate, 0.1% SDS at 50°C.; (2) employ a denaturing agent, such as formamide, duringhybridization for example, 50% (v/v) formamide with 0.1% BSA, 0.1%Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH6.5 with 750 mM sodium chloride/50 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC, 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA(50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at42° C. in 0.2×SSC and at 55° C. in 50% formamide, followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

Polypeptide Production

The polypeptides, derivatives or fragments thereof for use in themethods of the invention can be produced recombinantly and, if needed,chemically modified. Recombinant expression of a polypeptide requiresconstruction of an expression vector containing a polynucleotide thatencodes the polypeptide of interest (i.e., an SA2451 polypeptide,derivative or fragment thereof). Once a polynucleotide encoding thepolypeptide of interest has been obtained, the vector for the productionof the polypeptide of interest may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a polypeptide of interest by expressing a polynucleotideencoding said polypeptide are described herein.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequences of thepolypeptide of interest and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding a polypeptide of interestoperably linked to a promoter.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce the polypeptide of interest. Thus, the inventionincludes host cells containing a polynucleotide encoding a polypeptideof interest operably linked to a heterologous promoter.

A variety of host-expression vector systems may be utilized to expressthe polypeptides of interest. Such host-expression systems representvehicles by which the coding sequences of interest may be produced andsubsequently purified, but also represent cells which may, whentransformed or transfected with the appropriate nucleotide codingsequences, express the polypeptide of interest in situ. These includebut are not limited to microorganisms such as bacteria (e.g., E. coli,members of the Staphylococcus genus, such as S. aureus and S.epidermidis; members of the Lactobacillus genus, such as L. plantarum;members of the Lactococcus genus, such as L. lactis; members of theBacillus genus, such as B. subtilis; members of the Corynebacteriumgenus such as C. glutamicum; and members of the Pseudomonas genus suchas Ps. fluorescens.) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing coding sequencesof interest; yeast (e.g., Saccharomyces genus such as S. cerevisiae orS. pichia, members of the Pichia genus such as P. pastoris, members ofthe Hansenula genus such as H. polymorpha, members of the Kluyveromycesgenus such as K. lactis or K. fragilis, and members of theSchizosaccharomyces genus such as S. pombe) transformed with recombinantyeast expression vectors containing coding sequences of interest; insectcell systems infected with recombinant virus expression vectors (e.g.,baculovirus) containing coding sequences of interest; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingcoding sequences of interest; or mammalian cell systems (e.g., COS, CHO,BHK, 293, NSO, and 3T3 cells) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter) andcoding sequences of interest.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Eukaryotic modifications mayinclude glycosylation and processing (e.g., cleavage) of proteinproducts. Different host cells have characteristic and specificmechanisms for the post-translational processing and modification ofproteins and gene products. Appropriate cell lines or host systems canbe chosen to ensure the correct modification and processing of theforeign protein expressed. To this end, host cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Asused herein, the terms “polypeptide” or “an amino acid sequence of apolypeptide” includes polypeptides containing one or more amino acidshaving a structure of a post-translational modification from a hostcell, such as a yeast host.

Once a polypeptide of interest has been produced by recombinantexpression, it may be purified by different methods, for example, bychromatography (e.g., ion exchange, affinity, sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, thepolypeptides of interest may be fused or attached to heterologouspolypeptide sequences described herein or otherwise known in the art tofacilitate purification. Examples of such protein tags include, but arenot limited to, chitin binding protein (CBP), maltose binding protein(MBP), glutathione-S-transferase (GST), poly-histidine, hemagglutinin(HA) and polyanionic amino acids.

If desired, expression in a particular host can be enhanced throughcodon optimization. Codon optimization includes use of more preferredcodons. Techniques for codon optimization in different hosts are wellknown in the art.

Determination of Immunoreactive Derivatives and Fragments

The invention also extends to a method of identifying an immunoreactivederivative or fragments (collectively “altered polypeptides”) of anSA2451 polypeptide. This method essentially comprises generating aderivative or fragment of the polypeptide, administering the alteredpolypeptide to a mammal; and detecting an immune response in the mammal.Such response will include production of elements which specificallybind S. aureus and/or said polypeptide, derivative or fragment and/orhave a protective effect against S. aureus infection. Antibody titersand immunoreactivity against the native or parent polypeptide may thenbe determined by, for example, radioimmunoassay, ELISA, western blot orELISPOT.

Adjuvants

Adjuvants are substances that can assist an immunogen (e.g., apolypeptide, pharmaceutical composition containing a polypeptide) inproducing an immune response. Adjuvants can function by differentmechanisms such as one or more of the following: increasing the antigenbiologic or immunologic half-life; improving antigen delivery toantigen-presenting cells; improving antigen processing and presentationby antigen-presenting cells; and, inducing production ofimmunomodulatory cytokines (Vogel, Clinical Infectious Diseases 30(suppl. 3): S266-270, 2000). In one embodiment of the present invention,an adjuvant is used.

A variety of different types of adjuvants can be employed to assist inthe production of an immune response. Examples of particular adjuvantsinclude aluminum hydroxide; aluminum phosphate, aluminumhydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate adjuvant(AAHSA) or other salts of aluminum; calcium phosphate; DNA CpG motifs;monophosphoryl lipid A; cholera toxin; E. coli heat-labile toxin;pertussis toxin; muramyl dipeptide; Freund's incomplete adjuvant; MF59;SAF; immunostimulatory complexes; liposomes; biodegradable microspheres;saponins; nonionic block copolymers; muramyl peptide analogues;polyphosphazene; synthetic polynucleotides; IFN-γ; IL-2; IL-12; andISCOMS. (Vogel, Clinical Infectious Diseases 30 (suppl 3): S266-270,2000; Klein et al., 2000, Journal of Pharmaceutical Sciences 89:311-321;Rimmelzwaan et al., 2001, Vaccine 19:1180-1187; Kersten, 2003, Vaccine21:915-920; O'Hagen, 2001, Curr. Drug Target Infect. Disord. 1:273-286.)

Combination Vaccines

An SA2451 polypeptide, derivative or fragment thereof can be used alone,or in combination with other immunogens, to induce an immune response.Additional immunogens that may be present include one or more additionalS. aureus immunogens, one or more immunogens targeting one or more otherStaphylococcus organisms such as S. epidermidis, S. haemolyticus, S.warneri, S. pyogenes, or S. lugunensi, and/or one or more immunogenstargeting other infectious organisms including, but not limited to, thepathogenic bacteria H. influenzae, M. catarrhalis, N. gonorrhoeae, E.coli, S. pneumoniae, C. difficile, C. perfringens, C. tetani, bacteriaof the genuses Klebsiella, Serratia, Enterobacter, Proteus, Pseudomonas,Legionella, and Citrobacter.

In one embodiment, the additional immunogen is IsdB (also known asORF0657) or related polypeptides. Reference to an IsdB immunogen refersto an immunogen that produces a protective immune response thatrecognizes the IsdB protein in S. aureus. In different embodiments, theIsdB immunogen produces an immune response that recognizes IsdB presenton one or more of the following strains: COL, Becker, MW2, N315, Newman,USA300, MSA817, and Mu3. The ability of an IsdB immunogen to providedprotective immunity is illustrated in, for example, US Publication No.2006/0177462 (which is incorporated by reference herein in itsentirety).

In additional embodiments, the IsdB immunogen comprises a polypeptideregion, said region (a) is at least 90%, at least 94%, at least 95% orat least 99% identical to SEQ ID NO:6 or a fragment thereof (including,but not limited to, amino acids 42-486, 42-522 and 42-608 of SEQ IDNO:6); (b) differs from SEQ ID NO:6 or a fragment thereof (including,but not limited to, amino acids 42-486, 42-522 and 42-608 of SEQ IDNO:6) by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25 alterations, or up to 50 alterations;or (c) consists essentially or consists of SEQ ID NO:6 or a fragmentthereof (including, but not limited to, amino acids 42-486, 42-522 and42-608 of SEQ ID NO:6). Examples of alterations include amino acidsubstitutions, deletions, and insertions.

Reference to “consists essentially” of indicated amino acids indicatesthat the referred to amino acids are present and additional amino acidsmay be present. The additional amino acids can be at the carboxyl oramino terminus. In different embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additional amino acids arepresent. In preferred embodiments methionine is present at the aminoterminus; or methionine-glycine is present at the amino terminus.

In other embodiments, the one or more additional immunogens include, butare not limited to, ORF0657/ORF0190 hybrid polypeptides (InternationalPublication No. WO 05/009378 and US Publication No. 2006/0188515);ORF0688-related polypeptides; ORF0452-related polypeptides (USPublication 2008/131447); S. epidermidis ORF1319E-related polypeptides(WO 08/140632); S. epidermidis ORF2695E-related polypeptides (WO08/115415); ORF0912-related polypeptides; ORF1902-related polypeptides;sai-1-related polypeptides (International Publication No. WO 05/79315);ORF0594-related polypeptides (International Publication No. WO05/086663); ORF0826-related polypeptides (International Publication No.WO 05/115113); PBP4-related polypeptides (International Publication No.WO 06/033918); AhpC-related polypeptides and AhpC-AhpF compositions(International Publication No. WO 06/078680); PBP4-related polypeptides(WO06/033918); SACOL1902-related polypeptides (WO 10/062814);SACOL0912-related polypeptides (WO 10/062815); SA0024-relatedpolypeptides (WO 07/001361); SACOL 2451-related polypeptides;SACOL2412-related polypeptides (PCT/US11/43499), SACOL1789-relatedpolypeptides (PCT/US11/43274), SA2074-related polypeptides; S. aureustype 5 and type 8 capsular polysaccharides (Shinefield et al., 2002, N.Eng. J. Med. 346:491-496); collagen adhesin, fibrinogen bindingproteins, and clumping factor (Mamo et al., 1994, FEMS Immunol. Med.Microbiol. 10:47-54; Nilsson et al., 1998, J. Clin. Invest.101:2640-2649; Josefsson et al., 2001, J. of Infect. Dis.184:1572-1580); and polysaccharide intercellular adhesin and fragmentsthereof (Joyce et al., 2003, Carbohydrate Research 338:903-922).

Nucleic Acid Vaccine

The nucleic acid sequence of wild type full length SA2451 from S. aureussubsp. aureus COL is SEQ ID NO:3 (FIG. 1C). SEQ ID NO:3 or other nucleicacids that encode an SA2451 polypeptide of SEQ ID NO:1, derivative orfragment thereof can be introduced into a patient using vectors suitablefor therapeutic administration. Suitable vectors can deliver the nucleicacid into a target cell without causing an unacceptable side effect.Examples of vectors that can be employed include plasmid vectors andviral based vectors. (Barouch, 2006, J. Pathol. 208:283-289; Emini etal., International Publication No. WO 03/031588.)

Cellular expression is achieved using a gene expression cassetteencoding the desired polypeptide. The gene expression cassette containsregulatory elements for producing and processing a sufficient amount ofnucleic acid inside a target cell to achieve a beneficial effect.

Examples of viral vectors include first and second generationadenovectors, helper dependent adenovectors, adeno-associated viralvectors, retroviral vectors, alphavirus vectors (e.g., Venezuelan EquineEncephalitis virus vectors), and plasmid vectors. (Hitt a al., 1997,Advances in Pharmacology 40:137-206; Johnston et al., U.S. Pat. No.6,156,588; Johnston et al., International PCT Publication no. WO95/32733; Barouch, 2006, J. Pathol. 208:283-289; Emini et al.,International PCT Publication no. WO 03/031588.)

Adenovectors can be based on different adenovirus serotypes such asthose found in humans or animals. Examples of animal adenovirusesinclude bovine, porcine, chimpanzee, murine, canine, and avian (CELO).(Emini et al., International PCT Publication no. WO 03/031588; Collocaet al., International PCT Publication no. WO 05/071093.) Humanadenovirus include Group B, C, D, or E serotypes such as type 2 (“Ad2”),4 (“Ad4”), 5 (“Ad5”), 6 (“Ad6”), 24 (“Ad24”), 26 (“Ad26”), 34 (“Ad34”)and 35 (“Ad35”).

Nucleic acid vaccines can be administered using different techniques anddosing regimes (see, e.g., International Publication No. WO 03/031588and U.S. Pat. No. 7,008,791). For example, the vaccine can beadministered intramuscular by injection with or without one or moreelectric pulses. Electric mediated transfer can assist geneticimmunization by stimulating both humoral and cellular immune responses.Examples of dosing regimes include prime-boost and heterologousprime-boost approaches.

SA2451 Antibodies

An SA2451 polypeptide, derivative or fragment thereof can be used togenerate antibodies and antibody fragments that bind to SA2451 or to S.aureus. Such antibodies and antibody fragments can be used inpolypeptide purification, S. aureus identification, and/or intherapeutic treatment of S. aureus infection. In preferred embodiments,antibodies and/or antibody fragments thereof are administered to apatient in need thereof to provide passive immunity to S. aureus.

As used herein, the term “antibody” as used includes monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), single-chain Fvs (scFv) (including bi-specificscFvs), single chain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked Fvs (sdFv), and epitope-binding fragments of any of theabove, so long as they exhibit the desired biological activity. Inpreferred embodiments, antibodies of the invention are monoclonal. In amore preferred embodiment, the monoclonal antibodies used in the methodsof the invention are humanized or human antibodies.

As used herein, the term “monoclonal antibody” refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themonoclonal antibodies to be used in accordance with the presentinvention may be made by the hybridoma method first described by Kohlerand Milstein, Nature 256, 495 (1975), or may be made by recombinantmethods, e.g., as described in U.S. Pat. No. 4,816,567. The monoclonalantibodies for use with the present invention may also be isolated fromphage antibody libraries using the techniques described in Clackson etal. Nature 352: 624-628 (1991), as well as in Marks et al., J. Mol. Biol222: 581-597 (1991).

“Humanized” forms of non-human (e.g., murine) antibodies areimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from acomplementarity-determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. Thus, a humanized antibody is a recombinant protein in whichthe CDRs from an antibody from one species; e.g., a rodent antibody, istransferred from the heavy and light variable chains of the rodentantibody into human heavy and light variable domains. The constantdomains of the antibody molecule are derived from those of a humanantibody. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (see, e.g., Yamashita et al., 2007, Cytotech. 55:55;Kipriyanov and Le Gall, 2004, Mol. Biotechnol. 26:39 and Gonzales etal., 2005, Tumour Biol. 26:31).

Completely human antibodies may be desirable for therapeutic treatmentof human patients. Human antibodies can be made by a variety of methodsknown in the art including phage display methods described above usingantibody libraries derived from human immunoglobulin sequences. See U.S.Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645; WO98/50433; WO 98/24893 and WO 98/16654, each of which is incorporatedherein by reference in its entirety. Human antibodies can also beproduced using transgenic mice which are incapable of expressingfunctional endogenous immunoglobulins, but which can express humanimmunoglobulin genes. See, e.g., PCT publications WO 98/24893; EuropeanPatent No. 0 598 877; U.S. Pat. Nos. 5,916,771; and 5,939,598, which areincorporated by reference herein in their entireties.

In some embodiments, Fc engineered variants antibodies of the inventionare also encompassed by the present invention. Such variants includeantibodies or antigen binding fragments thereof which have beenengineered so as to introduce mutations or substitutions in the Fcregion of the antibody molecule so as to improve or modulate theeffector functions of the underlying antibody molecule relative to theunmodified antibody. In general, improved effector functions refer tosuch activities as CDC, ADCC and antibody half life (see, e.g., U.S.Pat. Nos. 7,371,826; 7,217,797; 7,083,784; 7,317,091; and 5,624,821,each of which is incorporated herein in its entirety).

There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM.The IgG and IgA classes are further divided into subclasses on the basisof relatively minor differences in the constant heavy region sequenceand function, e.g., humans express the following subclasses: IgG1, IgG2,IgG3, IgG4, IgA1, and IgA2. In preferred embodiments, the antibodies ofthe invention are IgG1.

Proper glycosylation can be important for antibody function (Yoo et al.,2002, J. Immunol. Methods 261:1-20; Li et al., 2006, Nature Biotechnol.24:210-215). Naturally occurring antibodies contain at least oneN-linked carbohydrate attached to a heavy chain (Yoo et al., supra).Additional N-linked carbohydrates and O-linked carbohydrates may bepresent and may be important for antibody function Id.

Different types of host cells can be used to provide for efficientpost-translational modifications including mammalian host cells andnon-mammalian cells. Examples of mammalian host cells include Chinesehamster ovary (Cho), HeLa, C6, PC 12, and myeloma cells (Yoo et al.,supra; Persic et al., 1997, Gene 187:9-18). Non-mammalian cells can bemodified to replicate human glycosylation (Li et al., 2006, NatureBiotechnol. 24:210-215). Glycoengineered Pichia pastoris is an exampleof such a modified non-mammalian cell (Li et al., supra).

Patient Population

A “patient” refers to a mammal capable of being infected with S. aureus.In one preferred embodiment, the patient is a human. In alternativeembodiments, the patient is a non-human mammal such as a dog or a cow. Apatient can be treated prophylactically or therapeutically. Prophylactictreatment provides sufficient protective immunity to reduce thelikelihood or severity of a S. aureus infection. Therapeutic treatmentcan be performed to reduce the severity of a S. aureus infection.

Prophylactic treatment can be performed using a pharmaceuticalcomposition containing a polypeptide, immunogen or antibody describedherein. Pharmaceutical compositions can be administered to the generalpopulation or to those persons at an increased risk of S. aureusinfection.

Those “in need of treatment” include those already with an infection, aswell as those prone to have an infection or in which a reduction in thelikelihood of infection is desired. Persons with an increased risk of S.aureus infection include health care workers; hospital patients;patients with weakened immunity; patients facing therapy leading to aweakened immunity (e.g., undergoing chemotherapy or radiation therapyfor cancer or taking immunosuppressive drugs); patients undergoingsurgery; patients receiving foreign body implants (such a catheter or avascular device); patients under diagnostic procedures involving foreignbodies; patients on renal dialysis and persons in professions having anincreased risk of burn or wound injury. As used herein, “weakenedimmunity” refers to an immune system that is less capable of battlinginfections because of an immune response that is not properlyfunctioning or is not functioning at the level of a normal healthyadult. Examples of patients with weakened immunity are patients that areinfants, young children, elderly, pregnant or a patient with a diseasethat affects the function of the immune system such as HIV or AIDS.

Foreign bodies used in diagnostic or therapeutic procedures includeindwelling catheters or implanted polymer device. Examples of foreignbody-associated S. aureus infections include septicemia/endocarditis(e.g., intravascular catheters, vascular prostheses, pacemaker leads,defibrillator systems, prosthetic heart valves, and left ventricularassist devices); peritonitis (e.g., ventriculo-peritoneal cerebrospinalfluid (CSF) shunts and continuous ambulatory peritoneal dialysiscatheter systems); ventriculitis (e.g., internal and external CSFshunts); and chronic polymer-associated syndromes (e.g., prostheticjoint/hip loosening, fibrous capsular contracture syndrome after mammaryargumentation with silicone prosthesis and late-onset endophtalmisisafter implantation of artificial intraocular lenses following cataractsurgery). (See, Hellmann and Peters, Biology and Pathogenicity ofStaphylococcus epidermidis, In: Gram Positive Pathogens, Eds. Fischettiet al., American Society for Microbiology, Washington D.C. 2000.)

Non-human patients that can be infected with S. aureus include cows,pigs, sheep, goats, rabbits, horses, dogs, cats, rats and mice.Treatment of non-human patients is useful in both protecting pets andlivestock (e.g. against Staph-related disease common to animals such asbovine mastitis) and evaluating the efficacy of a particular treatment.In addition to the obvious benefits of preventing, or reducing thelikelihood or severity of clinical manifestations of S. aureusinfections in vaccinated animals, additional benefits include thereduction of costs resulting from sick and underproductive livestockanimals to a farmer; the reduction in the need for quarantine measuresto a human or veterinary clinic by reducing the number of S. aureusinfected patients, and reduced need for repeated rigorousdecontamination of equipment and facilities; and a reduction of thenumber of S. aureus carriers in the human and animal populations, whichreduces their potential contamination and spread to others.

In an embodiment, a patient is treated prophylactically in conjunctionwith a therapeutic or medical procedure involving a foreign body. Inadditional embodiments, the patient is immunized at about 2 weeks, 1month, about 2 months or about 2-6 months prior to the procedure. Inanother embodiment, the patient is immunized prophylactically not inconjunction with a particular contemplated procedure. For vaccinations,boosters are delivered as needed. Additionally, patients treatedprophylactically may also receive passive immunotherapy byadministration of an antibody protective against S. aureus alone or inconjunction with vaccination.

Pharmaceutical Compositions

A further feature of the invention is the use of an SA2451 polypeptide,derivative or fragment thereof described herein (“immunogenic agent”),either alone or in combination with one or more additional antigens, ina composition, preferably an immunogenic composition or vaccine, fortreating patients with an S. aureus infection, reducing the progression,onset or severity of pathological symptoms associated with S. aureusinfection and/or reducing the likelihood of an S. aureus infection.Suitably, the composition comprises a pharmaceutically acceptablecarrier.

In some embodiment of the invention described above, the pharmaceuticalcompositions are used in human patients. In alternative embodiments, thepharmaceutical compositions are used in non-human patients.

A “pharmaceutically-acceptable carrier” is meant to mean a liquidfiller, diluent or encapsulating substance that may be safely used insystemic administration. Depending upon the particular route ofadministration, a variety of pharmaceutically acceptable carriers, wellknown in the art may be used. These carriers may be selected from agroup including sugars, starches, cellulose and its derivatives, malt,gelatine, talc, calcium sulfate, vegetable oils, synthetic oils,polyols, alginic acid, phosphate buffered solutions including phosphatebuffered saline, emulsifiers, isotonic saline, and pyrogen-free water.In particular, pharmaceutically acceptable carriers may containdifferent components such as a buffer, sterile water for injection,normal saline or phosphate-buffered saline, sucrose, histidine, saltsand polysorbate. Terms such as “physiologically acceptable”, “diluent”or “excipient” can be used interchangeably.

The above compositions may be used as therapeutic or prophylacticvaccines. Accordingly, the invention extends to the production ofvaccines containing as active ingredients one or more of the immunogenicagents of the invention. Any suitable procedure is contemplated forproducing such vaccines. Exemplary procedures include, for example,those described in New Generation Vaccines (1997, Levine et al., MarcelDekker, Inc. New York, Basel Hong Kong), which is incorporated herein byreference.

A polypeptide of the invention can be fused or attached to animmunogenic carrier. Useful carriers are well known in the art andinclude for example: thyroglobulin; albumins such as human serumalbumin; toxins, toxoids or any mutant crossreactive material (CRM) ofthe toxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli,Staphylococcus, and Streprococcus; polyamino acids such aspoly(lysine:glutamic acid); influenza; Rotavirus VP6, Parvovirus VP1 andVP2; hepatitis B virus core protein; hepatitis B virus recombinantvaccine and the like. Alternatively, a fragment or epitope of a carrierprotein or other immunogenic protein may be used. For example, a peptideof the invention can be coupled to a T cell epitope of a bacterialtoxin, toxoid or CRM. In this regard, reference may be made to U.S. Pat.No. 5,785,973, which is incorporated herein by reference.

Administration/Methods of Treatment

An SA2451 polypeptide, derivative or fragment thereof (alone or incombination with one or more immunogens) or an antibody described hereincan be formulated and administered to a patient using the guidanceprovided herein along with techniques well known in the art. Guidelinesfor pharmaceutical administration in general are provided in, forexample, Vaccines Eds. Plotkin and Orenstein, W.B. Sanders Company,1999; Remington's Pharmaceutical Sciences 20^(th) Edition, Ed. Gennaro,Mack Publishing, 2000; and Modern Pharmaceutics 2^(nd) Edition, Eds.Banker and Rhodes, Marcel Dekker, Inc., 1990.

Accordingly, the invention provides a method for inducing a protectiveimmune response in a patient against an S. aureus infection comprisingthe step of administering to the patient an immunologically effectiveamount of any of the vaccines or pharmaceutical compositions describedherein. In one embodiment of this aspect of the invention, the patientis a human. In alternative embodiments, the patient is a non-humanmammal.

Also provided by the invention is a method for treating S. aureusinfection, or for treating any pathological condition associated with S.aureus infection, the method comprising the step of administering to thepatient an immunologically effective amount of any of the vaccines orpharmaceutical compositions described herein. In one embodiment of thisaspect of the invention, the patient is a human. In alternativeembodiments, the patient is a non-human mammal.

Vaccines and/or antibodies can be administered by different routes suchas subcutaneous, intramuscular, intravenous, mucosal, parenteral ortransdermal. Subcutaneous and intramuscular administration can beperformed using, for example, needles or jet-injectors.

In some embodiments, the vaccines and/or antibodies of the invention canbe formulated in or on virus-like particles (see, e.g., InternationalPublication Nos. WO94/20137; WO96/11272; U.S. Pat. Nos. 5,985,610;6,599,508; 6,361,778), liposomes (see, e.g., U.S. Pat. No. 5,709,879),bacterial or yeast ghosts (empty cells with intact envelopes; see, e.g.,International Publication WO 92/01791, US Publication No. 2009/0239264and 2008/0003239, U.S. Pat. No. 6,951,756), and outer membrane vesiclesor blebs (de Moraes et al., 1992, Lancet 340: 1074 and Bjune et al.,1991, Lancet 338: 1093).

The compositions described herein may be administered in a mannercompatible with the dosage formulation, and in such amount as isimmunogenically-effective to treat and/or reduce the likelihood of S.aureus infection. The dose administered to a patient, in the context ofthe present invention, should be sufficient to effect a beneficialresponse in a patient over time such as a reduction in the level of S.aureus, or to reduce the likelihood of infection by S. aureus. Thequantity of the immunogenic agent(s) to be administered may depend onthe subject to be treated inclusive of the age, sex, weight and generalhealth condition thereof. In this regard, precise amounts of theimmunogenic agent(s) required to be administered will depend on thejudgment of the practitioner. In determining the effective amount of theimmunogenic agent to be administered in the treatment or prophylaxisagainst S. aureus, the physician may evaluate circulating plasma levels,progression of disease, and the production of anti-S. aureus antibodies.In any event, suitable dosages of the immunogenic agents of theinvention may be readily determined by those of skill in the art. Suchdosages may be in the order of nanograms to milligrams of theimmunogenic agents of the invention.

Suitable dosing regimens are preferably determined taking into accountfactors well known in the art including age, weight, sex and medicalcondition of the patient; the route of administration; the desiredeffect; and the particular compound employed. The vaccine and/orantibody composition can be used in multi-dose formats. It is expectedthat a dose for a vaccine composition would consist of the range of 1.0μg to 1.0 mg total polypeptide. In different embodiments of the presentinvention, the dosage range is from 5.0 μg to 500 μg, 0.01 mg to 1.0 mg,or 0.1 mg to 1.0 mg. When more than one polypeptide is to beadministered (i.e., in combination vaccines), the amount of eachpolypeptide is within the described ranges.

It is expected that a dose for a passive immunity composition of theinvention would consist of the range of 1 μg/kg to 100 mg/kg ofantibody. In different embodiments, of the present invention, the dosagerange is from 1 μg/kg to 15 mg/kg, 0.05 mg/kg to about 10 mg/kg, 0.5mg/kg to 2.0 mg/kg, or 10 mg/kg to 50 mg/kg.

The timing of doses depends upon factors well known in the art. Afterthe initial administration one or more additional doses may beadministered to maintain and/or boost antibody titers.

For combination vaccinations, each of the polypeptides can beadministered together in one composition or separately in differentcompositions. An SA2451 polypeptide described herein is administeredconcurrently with one or more desired immunogens. The term“concurrently” is not limited to the administration of the therapeuticagents at exactly the same time, but rather it is meant that the SA2451polypeptides described herein and the other desired immunogen(s) areadministered to a subject in a sequence and within a time interval suchthat the they can act together to provide an increased benefit than ifthey were administered otherwise. For example, each therapeutic agentmay be administered at the same time or sequentially in any order atdifferent points in time; however, if not administered at the same time,they should be administered sufficiently close in time so as to providethe desired therapeutic effect. Each therapeutic agent can beadministered separately, in any appropriate form and by any suitableroute.

All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing methodologies and materials thatmight be used in connection with the present invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Having described preferred embodiments of the invention with referenceto the accompanying drawings, it is to be understood that the inventionis not limited to those precise embodiments, and that various changesand modifications may be effected therein by one skilled in the artwithout departing from the scope or spirit of the invention as definedin the appended claims.

The following examples illustrate, but do not limit the invention.

Example 1 Cloning and Expression of Antigen

Genomic DNA was purified from S. aureus COL strain MB5393 and used as atemplate for PCR. The SA2451 gene (SEQ ID NO:3) was amplified by PCR ina 50 μL volume reaction prepared in duplicate. Each reaction mixturecontained 250 ng genomic DNA, 125 ng each forward(CACCATGAAGAAAATTAAATATATACTTGTCGTGTTTGTCTTATCGC; SEQ ID NO:4) andreverse (TCAGTGGTGGTGGTGGTGGTGGTGGTGCTTATGACCACCTTTCTGTTTA; SEQ ID NO:5)primer, 1 μL 10 mM dNTPs, 2.5 units of native Pfu polymerase and 1× Pfubuffer (Stratagene, La Jolla Calif.). The thermocycling conditions wereas follows: one cycle of 94° C. for 5 minutes; 30 cycles of 94° C. for45 seconds, 56° C. for 45 seconds, 72° C. for one minute; one cycle of72° C. for 10 minutes. The amplified DNA sequence (966 bp) was digestedwith the appropriate restriction enzymes and gel-purified using GENECLEAN II® (QBIOgene, Carlsbad, Calif.) according to the manufacturer'sdirections. The DNA was ligated into the pENTR/Sd/D vector using theNdeI/Bpu1102I sites that had been engineered into the PCR primers andintroduced into E. coli NovaBlue competent cells (EMD Biosciences).

The SA2451 gene (SEQ ID NO:3) was cloned into an expression vector(pET-28b) that would facilitate either a carboxy or amino his tag to beadded to the expressed protein. E. coli competent cells were transformedwith the vector containing a nucleotide sequence encoding SA2451 ORFwith a histidine tag and grown on LB plates containing ampicillin (100μg/mL) and chloramphenicol (34 ng/ml). To test for expression of SA2451,an isolated colony was inoculated into 5 mL of liquid LB, 1% glucose,100 μg/ml ampicillin, and incubated at 37° C. or 25° C., 250 rpm, untilthe OD₆₀₀ was between 0.5 to 1.0. A 1.0 ml culture volume of cells wassubjected to centrifugation and resuspended in lysis buffer. Themixtures were held on ice for 5 minutes and subsequently sonicated threetimes for ten seconds, each with cooling in between. To obtain “soluble”and “insoluble” fractions the mixture was centrifuged at 13,000 rpm forfifteen minutes at 4° C. The supernatant was designated “soluble” andthe pellet was resuspended in lysis buffer and designated “insoluble.”

Expression of SA2451 (C-Histag with glycine cap) was analyzed byCoomassie staining of SDS-PAGE gels run under reducing and denaturingconditions. The gels were stained with Bio-Safe Coomassie, a CoomassieG250 stain (BIO-RAD) according to the manufacturer's protocol. Westernblot was performed and the signal was detected by anti-His mAb (EMDSciences). A 36 kDa protein was specifically detected by both Coomasiestaining and Western blot in lysates.

Direct scale-up of the above small scale procedure into stirred tankfermenters (30 liter scale) with a 20 liter working volume was achieved.Inoculum was cultivated in a 250 mL flask containing 50 mL of LB medium(plus ampicillin) and inoculated with 1 mL of frozen seed culture andcultivated for 6 hours. One mL of this seed was used to inoculate a 2liter flask containing 500 mL of LB medium (plus ampicillin) andincubated for 16 hours. A large scale fermenter (30 liter scale) wascultivated with 20 liters of LB medium (plus ampicillin). Thefermentation parameters of the fermenter were: pressure=5 psig,agitation speed=300 rpm, airflow=7.5 liters/minute and temperature=25°C. Cells were incubated to an optical density (OD) of 1.3 opticaldensity units at a wavelength of 600 nm. Cells were harvested bylowering the temperature to 15° C., concentrated by passage through a500KMWCO hollow fiber cartridge, and centrifuged at 8,000 times gravityat 4° C. for 20 minutes. Supernatants were decanted and the recombinantE. coli wet cell pellets were frozen at −70° C.

Frozen recombinant E. coli cell paste (56 grams) was thawed andresuspended in 565 mL of IMAC Buffer A (50 mM NaCl, 5 mM Imidazole, 20mM Tris, 125 mM Brij-35, 10 mM TritonX-100, 5 mM Tween 20, pH 7.5;BSB-protease inhibitor cocktail). A lysate was prepared with a PANDAcell homogenizer (Niro Soavi). The lysate was clarified bycentrifugation at 30,000×g for 60 minutes at 4° C. using a Beckman JA-21centrifuge with a JA-18 rotor. The supernatant was added to a 10 mlcolumn gravity packed with Ni Sepharose 6 Fast Flow resin (GEHealthcare) connected to an AKTA FPLC previously equilibrated with 10 CVof IMAC Buffer A. Following a 10CV wash of the column with 2% IMACBuffer B (IMAC Buffer A+1 M Imidazole), bound protein was eluted in 5 mlfractions at 5 ml/min using a linear imidazole gradient of 2-100% IMACBuffer B. Fractions containing protein were analyzed by SDS-PAGEfollowed by coomassie staining and Western Blotting using anti-Hisantibody. The fractions were pooled based on SDS-PAGE data and thepooled volume was dialyzed against S Buffer A (20 mM MES, pH 6.5, 20 mMKCl) overnight at 4° C. A HiTrap SPFF column (2×5 ml cartridges, GEHealthcare) was connected to an ÄKTA FPLC and was equilibrated with 10CV of S Buffer A after being charged with S Buffer B (20 mM MES, pH 6.5,1 M KCl). The pool was loaded onto the column (GE Healthcare) at a flowrate of 5 ml/min, and the column was washed with S Buffer A at a flowrate of 5 ml/min. Following a 2% S Buffer B wash, bound protein waseluted in 5 ml size fractions at 5 ml/min using a linear KCl gradient of2-100% S Buffer B. Protein containing fractions were analyzed bySDS-PAGE followed by

Coomassie-staining or Western blotting (anti-His antibody). SA2451containing fractions were pooled and dialyzed against a final buffer (10mM MOPS, 150 mM NaCl, pH 7.0) overnight at 4° C. The protein solutionwas concentrated to 0.2 mg/mL and processed to remove endotoxin andfrozen at −70° C. The protein was thawed and adsorbed onto aluminumhydroxyphosphate adjuvant (“Merck Aluminum Adjuvant” or “MAA”) at afinal concentration of 0.1 mg/mL. This protein was used inimmunogenicity and protection studies.

Example 2 Immunogenicity Studies

Balb/c mice, at 3-5 weeks of age, were immunized on days 0, 7 and 21intramuscularly with a formulation of His-tagged SA2451 (20 μg perinjection) adsorbed onto MAA. Negative control mice were injected withbovine serum albumin (BSA) formulated on MAA, or not immunized. Immunesera from the mice was tested for reactivity to SA2451 by ELISA and wasfound to contain high antibody titers to the antigen. Negative controlantisera did not have antibody titers to SA2451 (Data not shown). Immunesera was also analyzed for reactivity to S. aureus cells by Flowcytometry. Results show the antigen was immunogenic in Balb/c mice.

Example 3 Murine Lethal Challenge Model

In two independent experiments, Balb/c mice immunized with either SA2451or BSA formulated on MAA (20 per group) or unimmunized (see EXAMPLE 2)were challenged with S. aureus Becker (8×10⁸ CFU/mouse) injected via thetail vein two weeks after immunization. Mice were monitored for survivalfor a period of 10 days post challenge. At the end of the firstexperiment (#MSE116), 8 mice survived in the SA2451polypeptide-immunized group, compared to 2 surviving in the BSA controlgroup. Thus, the group of mice immunized with SA2451 had a significantlyenhanced survival rate compared to the negative control mice immunizedwith BSA (p=0.0027). In the second experiment (#MSE120), 12 micesurvived in the SA2451 polypeptide-immunized group, compared to 5surviving in the BSA control group. Results are shown for the twoexperiments in FIG. 3 and survival curves are compared in FIG. 4.Similar to the first experiment, mice immunized with SA2451 had asignificantly enhanced survival rate compared to the negative controlmice immunized with BSA (p=0.0250).

Example 4 Rhesus Macaque Immunogenicity

Rhesus macaques (n=3) were immunized with 50 μg of SA2451 formulated onMerck alum adjuvant (MAA), intramuscularly, on day 0. Animals were bledat designated time points and the immune response to the antigenmeasured using an ELISA type assay, with a Mesoscale instrument (as perthe manufacturer's instructions). The titers are measured in RLU(relative luminescence units). The monkeys' titers were increased by 4weeks post immunization. At eight weeks post immunization, the animalswere challenged via the intravenous route with a sublethal dose of S.aureus Becker (2×10^9 CFU). Measurement of immune titers post challengeindicated that two of three monkeys had increased Ab titers to thebacteria, indicating a memory response to the antigen on the bacteria.

What is claimed is:
 1. A method of inducing an immune response in apatient against Staphylococcus aureus infection comprising administeringan immunologically effective amount of the polypeptide of SEQ ID NO: 1,a pharmaceutically acceptable carrier and an adjuvant.
 2. The method ofclaim 1 wherein the patient is human or a non-human mammal.
 3. Themethod of claim 2 wherein the patient has weakened immunity, hasreceived a foreign body implant or is on renal dialysis.
 4. The methodof claim 3 wherein the patient that has weakened immunity has a HumanImmunodeficiency Virus (HIV) infection or Acquired Immune DeficiencySyndrome (AIDS).
 5. The method of claim 3 wherein the foreign bodyimplant is a catheter, a vascular device, pacemaker leads, defibrillatorsystems, or prosthetic heart valve.
 6. The method of claim 1, whereinthe composition further comprises one or more additional S. aureusantigens.